Unlike the GPCR-based vision, smell and tastes, the molecular mechanisms of hearing, touch, and blood-pressure or systemic-osmolarity sensing are unknown. How ion channels receive mechanical forces remain obscure, except for bacterial channels MscL, MscS. We are using the rat TRP channel V4 subtype, to help fill this knowledge gap. Against expectation, we have now shown TRPV4's direct and instant response to membrane stretch under Xenopus oocyte patch clamp. We have also developed yeast as a new arena to dissect TRPV4. Our work is challenging the dominant TRPV4's osmotic- gating model. We plan to define the force-sensing domain within TRPV4, using strategic peptide insertions, truncations and chimeras. We will also test the force-from-lipid theory by examining the effects of membrane-intercalating compounds and by mutating putative lipid-anchoring amino acids. 2008-10 found TRPV4 gain-of-function (GOF) mutations to cause human bone-development pathologies (brachyolmia, metatropic dysplasia, etc.). To characterize and understand the molecular defects caused by these disease alleles, we will elucidate TRPV4's additional gating mechanisms, and will examine all seven human GOF alleles in molecular detail with improved electrophysiological techniques and yeast-based phenotyping.